Because of evidence that relative metabolic activity (cycling cells compared to non cycling cells) and metabolic heterogeneity (a range of intracellular pH as well as cell cycle distribution) can influence cell sensitivity to chemotherapy, we first propose to examine the effect of the hormones insulin, thyroxine and estrogen on the metabolism of two human breast cell lines, MCF-7 (estrogen receptor positive) and MDA MB231 (estrogen receptor negative). Metabolic measurements will include glucose transport and utilization, amino acid uptake, lactic acid production, cellular ATP, and cellular respiration. Growth response measurements will include growth curves, viability and clonogenicity; and flow cytometric analysis of DNA, RNA and protein content, chromatin structure (growth fraction), as well as esterase activity and the intracellular pH distribution which is used as direct evidence of relative metabolic heterogeneity. The influence of metabolic and growth characteristics on cell sensitivity to methotrexate, 5-FU and adriamycin will then be assessed using these metabolic and growth response measurements as a guide to hormonal manipulation. Since sensitization of MCF-7 cells to methotrexate by insulin has been observed, this combination and other synergistic combinations will be examined in normal mice for survival and bone marrow toxicity (CFU-C/CFU-S). In addition, synergistic combinations will be used in the MCF-7 nude mouse tumor model system enabling concurrent measurements to be made of the tumor and bone marrow response. Finally, the influence of metabolic modification by individual hormones on drug transport will be measured to distinguish transport from other metabolic effects in synergistic hormone-drug combinations. The objective of these studies is to find ways to activate the metabolic pathways on which cells are both dependent and vulnerable to active cytotoxic agents, so producing metabolically homogeneous cell populations that are more sensitive. Clinical studies could follow that transiently modify tumor cell metabolism just prior to chemotherapy.